High molecular biopolymers of the carbohydrate nature of the plant Prunus domestica L. fruits

. This paper describes the technology developed by us for obtaining some physiologically active polysaccharides (pectic substances and hemicellulose) from domestic plum. The isolated products were identified by IR spectroscopy, which made it possible to determine their qualitative characteristics. The dried fruits of Prunus domestica L. were analyzed for the content of low molecular weight substances, then polysaccharides were isolated from plant materials, hydrolyzed, acetylated, and the monosaccharide composition of each polysaccharide was studied by a combination method of gas chromatography and mass spectrometry. A thermal analysis of the obtained polysaccharides was also carried out to determine their thermal stability.

It is known that the fruits of Prunus domestica L. have a mild laxative effect on the gastrointestinal tract (GIT) and are not addictive [15][16][17].The laxative property of fruits is determined by the content of natural biopolymers in them -polysaccharides of various chemical structures.Previously, we found that domestic plum growing on the territory of Uzbekistan contains substances related to dietary natural fibers -water-soluble polysaccharides, pectin substances and hemicellulose [18][19][20][21][22][23].
For structural characterization and deeper understanding, including the explanation of the biological activity of polysaccharides, it is necessary to determine their monosaccharide composition.In addition, the results of such analyzes help to conduct extensive biological studies involving polysaccharides and determine their biological activity.Monosaccharide composition assays are also useful for quality control of functional polysaccharides.The process is usually carried out by hydrolysis of polysaccharides with subsequent detection of released monosaccharides or their derivatives.This paper presents the conditions and results of a preliminary gas chromatographic/mass spectrometric (GC/MS) analysis of dried plum fruits, preparation of plant materials, and the production of the above polysaccharides in order to determine the monosaccharide composition by GC/MS.

Materials and methods
For the experiments we used plum fruits Prunus domestica L., collected during the period of full ripening.
Studies on the preliminary chromato-mass-spectrometric analysis of low molecular weight substances in dried raw materials were performed on an Agilent 8890 GCSystem (USA) gas chromatograph with split and splitless evaporators, which was used together with a 5977BGC/MSD mass spectrometer (USA) in the Scan and electron impact ionization.
Thermal studies were carried out using a synchronous thermal analyzer "Netzsch STA 409 PC Luxx", in the temperature range of 25-370 °C, with a heating rate of 5 K per minute, in aluminum crucibles.

Results
Drying of the sorted fruits, laid out in a thin layer on a layer of aluminum foil, was carried out by infrared drying in an oven ShS-80-01SPU at a temperature of 40-500C with periodic ventilation.As a result of using the method of infrared drying of raw materials, high-quality prunes were obtained with a characteristic dark color, texture, pulp, smell and taste.The mass distribution of dried fruits was as follows: peeled pulp (91.48%), stones (7.54%), substandard fruits (0.83%) and stalks (0.15%).The bulk density of fruits was 1.09 kg/l.
Sample preparation was performed as follows: the pulp of the dried fruit of Prunus domestica L. weighing about 1 g was crushed, poured with hexane in a volume of ≈ 5 ml, and left overnight.The next day was sonicated in Normal mode, at room temperature, for 1 minute.The obtained hexane extract was filtered through an Agilent membrane (USA) with a nominal pore size of 0.2 μm and injected into the column of the instrument (Figure 1).This procedure was repeated with chloroform (Figure 2).To obtain polysaccharides, the pulp of the dried fruits of the domestic plum Prunus domestica L. was used.
Pectin substances (PS) were isolated from the meal (remaining after the removal of water-soluble extractive substances) by double extraction with an aqueous solution of a mixture of 0.5% solutions of oxalic acid and ammonium oxalate (ratio 1:1), with a total hydromodulus of 1:3 (1:2 and 1:1) for 4 hours at 70°C.The resulting extract was filtered from the remains of plant material, dialyzed in a cellulose bag against distilled water with constant stirring, and concentrated under vacuum in a rotary evaporator (60-65°C) or a vacuum evaporator to obtain 1/10 of the original volume.The purified PS fraction was isolated from the resulting concentrate by precipitation with 96% ethanol in a ratio of 1:3-5.PS was filtered off, dried at a temperature not exceeding 60°С, and crushed.
Hemicellulose (HC) was isolated from the meal remaining after the production of PS.HC was obtained by three times extraction with a 5% aqueous solution of sodium hydroxide with a total hydromodulus of 1:4 (1:2; 1:1 and 1:1) and at room temperature for 3.5 hours.The extract was filtered from the remains of plant materials, neutralized with a 5% HcL aqueous solution, the formed salts were removed using a PallCorporation (USA) membrane at a pressure of 4-5 atm at room temperature and concentrated under vacuum in a rotary evaporator or vacuum evaporator to obtain 1/10 of the initial volume.The purified HC fraction was isolated from the resulting concentrate by precipitation with 96% ethanol in a ratio of 1:3-5.HC was filtered off, dried at a temperature not exceeding 60°C, and crushed.
Figure 3 shows the IR spectra of pectin (upper) and HZ (lower).Further, to obtain monosaccharide fragments of polysaccharides, an acid hydrolysis reaction was carried out: 3 ml of 2 M trifluoroacetic acid (TFA) (C2HF3O2) was poured into a polysaccharide sample in an amount of 10 mg and heated to 110°C for 4 h.TFA was removed by adding 15 ml of methanol (CH3OH).In the presence of methanol, TFA volatilizes and the remaining mixture is concentrated.
Upon completion of acid hydrolysis, the acylation reaction was carried out by the pyridine-acetic anhydride method: 10 mg of hydroxylamine hydrochloric acid ((NH3OH)Cl), 8 mg of inositol (C6H12O6), and 0.5 ml of pyridine (C5H5N) were added to the container with the concentrate.The mixture was heated to 90°C for 30 minutes and cooled to room temperature.Added acetic anhydride ((CH₃CO)₂O) in the amount of 0.5 ml and reheated to 90°C for 30 minutes.Then the mixture was centrifuged (DLAB, model D2012Plus, China), the supernatant was separated from the precipitate, followed by drying with an inert gas (N2), poured into 0.5 ml of chloroform, and analyzed in an Agilent 5977B chromato-mass spectrometer (Figures 3, 5, 7 and 8).
Chromatography conditions:  Column type OV-1701. Detector temperature 270°C. Injector temperature 250°C. Carrier gasesnitrogen N2 (50 ml/min), hydrogen H2 (50 ml/min) and oxygen O2 (500 ml/min).The device data were processed according to formulas (1) and ( 2).The resulting chromatograms are shown in Figures 4 and 5.  Thermoanalytical studies of polysaccharides were carried out on a Netzsch Simultaneous Analyzer STA 409 PG instrument (Germany), with a K-type thermocouple (LowRGSilver) and aluminum crucibles.All measurements were carried out in an inert nitrogen atmosphere with a nitrogen flow rate of 50 ml/min.The temperature range of measurements was 25-370°C, the heating rate was 5K/min.The amount of sample per measurement is 5-10 mg.The measuring system was calibrated with a standard set of substances KNO3, In, Bi, Sn, Zn.
The GC/MS device in the chloroform extract revealed only one substance, isolongifolan-8-ol (C15H26O, detection time 25.495 min, peak area 601075) with a molecular weight of 222.37.
The yield of PV from 1 kg of plant material was 3.3 g, and HC was 77.1 g.The IR spectra of HP and HC samples contain all vibrations corresponding to the characteristic vibration bands of polysaccharides ~1730÷1720 cm -1 , which corresponds to the interval 1740-1700 cm -1 and indicates the presence of non-esterified carboxyl groups listed in Table 1.
Table 1.The main vibrational frequencies (cm -1 ) found in the IR spectra of PS and HC isolated from Prunus domestica L.    On the curve of the TG derivatogram, the sum of polysaccharides up to 176.3°C, the weight loss was 4.5%, then, above this temperature, they begin to decompose at a rate of 2.5% / min.According to the DSC curve at 192°C, a pronounced maximum is observed, associated with the above indicated rate of mass change (Figure 8).
Thermal analysis of polysaccharides showed that the decomposition of substances begins at 295°C with a maximum rate.
When processing the obtained experimental information, data mining methods [30-37] were used, the effectiveness of which has been proven in data processing in hydromechanics [38,39], in processing data on farms in the Republic of Uzbekistan [40], in assessing health risk factors for highly qualified athletes [41], when processing data obtained as a result of studying the composition and technology of drying the fruits of the medicinal plant Capparis spinosa L. [42], as well as used in the educational process in the preparation of students and undergraduates of pharmaceutical higher educational institutions in the subject "Information technology and mathematical modeling processes" [43].

Conclusion
Preliminary chromatographic analysis of low molecular weight substances of dried fruits of Prunus domestica L. showed that infrared drying does not stimulate the formation of toxic substances.
The preparation of sugar aldonitrile acetates using the acetylation reaction provides primary information on the monosaccharide composition of plant polysaccharides.To fully describe the chemical structures and ascertain a correlation between the biological activity and the chemical structure of these natural substances, additional analyzes using the methylation reaction are required.
The polysaccharides of Prunus domestica L. showed thermal stability up to 176.3°C, based on these results, it can be concluded that the storage of substances at room temperature does not damage their nativeness anymore.